number of reasons. These included the fact that the chain length of naturally occurring DNA was much greater than for proteins.Developments did come in the form of RNA as it doesn’t share many of the drawbacks associated with DNA sequencing. RNAases with basespecificity were known and consequently
alanine tRNA in1965 became the first nucleic acid to be sequenced. Sinsheimer, R.L.(1959) states that the first DNA molecule purified to homogeneity was thegenome of bacteriophage X174. Another major breakthrough came with
the discovery of type II restriction enzymes by Hamilton smith and histeam of scientists. These enzymes had the ability to recognise and cleaveDNA at specific short nucleotide sequences. The restriction enzymes thenbecame used as a tool for the cutting of large DNA molecules into anumber of small pieces that could, using gel electrophoresis or 2Dchromatography be separated by size. The separation method used wascritical in relation to the size of the fragments that could be analysed. These methods weren’t able to determine complete gene sequences butimportant regulatory signals were sequenced for e.g. the
operon and phage lambda.Gel-based DNA sequencing methods became of importance during 1975with the “plus and minus” method of DNA sequencing. Herepolyacrylamide gels were used to separate the products of primedsynthesis by DNA polymerases. DNA polymerase reactions extended aprimer to a single stranded DNA template. Problems were associated withthis method i.e. no bands are produced for positions internal to runs but in1977 Sanger developed “the dideoxy method”. The use of gel-basedsequencing methods accelerated the rate of DNA sequencing.Improvements in the useful read length of dideoxy sequencing cameabout by using narrower gels and replacing
P labelling of DNA with
Single and multi gene disease traits
Since the 1950’s genetics has become an important part of medicalresearch and practice with single defective genes being the focus of thefirst studies carried out. The basis of modern genetics originated from theAustrian monk Gregor Mendel and his laws of inheritance. Single traitdisease include sickle cell anaemia, Duchenne muscular dystrophy etc.while Williams E. Evans (2004) states that the defective gene coding for TPMT arises from individuals caring both copies of the gene, occurs in onein 300 patients. TPMT results in the accumulation of the active compoundsof purine drugs. This can have a potentially fatal bone marrow reactionresulting in the lowering of white blood cell count.